Tissue adhesive

ABSTRACT

This invention relates to a tissue adhesive used in mammals for among other things to connect tissue or organs, in sealing wounds, stopping bleeding and healing wounds. The adhesive comprises specified amounts of factor XIII, fibrinogen, cold-insoluble globulin and albumin and plasminogen-activator inhibitor or plasmin inhibitor.

This application is a continuation-in-part application of U.S. application Ser. No. 118,656 filed Feb. 4, 1980 U.S. Pat. No. 4,298,598.

The invention relates to a tissue adhesive based on human or animal proteins containing factor XIII and fibrinogen.

It has long been known to use blood coagulating substances to stop bleeding and for sealing wounds. According to the first proposals of this kind, fibrin tampons and fibrin platelets, respectively, were used. During the Second World War, tissue adherence by means of blood plasma was suggested.

In recent times, a tissue adhesive based on fibrinogen and factor XIII for seamless interfascicular nerve transplantations in animal experiments has been described by H. Matras et al. in "Wiener Medizinische Wochenschrift", 1972, page 517.

A further study was carried out by Spangler et al. in "Wiener Klinische Wochenschrift", 1973, pages 1 to 7. Also there, the possibility was shown in animal experiments of carrying out a tissue adherence with the aid of fibrinogen as a cryoprecipitate and thrombin.

These known preparations have not yet proven to be satisfactory since they do not sufficiently meet the demands required of a tissue adhesive, i.e.

(a) high straining capacity of the adherences and wound sealings as well as safe and permanent blood stopping, i.e. good adhering capacity of the adhesive to the wound and tissue surfaces, as well as high internal strength of the same,

(b) controllable durability of the adherences in the body,

(c) complete absorbability of the adhesive in the course of the wound healing process,

(d) properties which stimulate wound healing.

This may partly be due to the fact that, in the known preparations, the coagulation factors necessary for stopping bleeding have not been present in an optimal proportion to one another, and also to the fact that the fibrinolytic activity in the area of adherence has not been sufficiently under control. Premature dissolutions of the tissue adherences frequently occured due to enzymatic influence.

The invention is aimed at avoiding these disadvantages and difficulties and has as its object to provide a tissue adhesive on the basis of fibrinogen and factor XIII of human or animal origin, which adhesive meets the requirements initially set out.

The invention is based on the finding that it is necessary to provide a certain ratio of fibrinogen to factor XIII and that the fibrinolytic activity is inhibited by the presence of an inhibitor in a concentration sufficient for the prevention of fibrinolysis.

The tissue adhesive according to the invention comprises plasma proteins of human or animal origin, which plasma proteins contain: factor XIII in an amount of at least 7 units/ml, fibrinogen in an amount of at least 70 mg/ml, the ratio of factor XIII to fibrinogen, expressed in units of factor XIII per gram of fibrinogen, amounting to at least 100; cold-insoluble globulin (CIG) and albumin, the ratio of fibrinogen to cold-insoluble globulin to albumin being 60 to 98:0.5 to 20:0 to 15; plasminogen-activator-inhibitor or plasmin-inhibitor selected from the group consisting of aprotinin, C1-inactivator, α₂ -antiplasmin, α₂ -macroglobulin, α₁ -antitrypsin, epsilonaminocaproic acid and tranexamic acid, the concentration being such that fibrinolysis is prevented, wherein the tissue adhesive as determined according to the SDS-polyacrylamide-gel-electrophoresis method is capable of complete cross-linking of the fibrin-γ-chains after 3 to 5 minutes of incubation and of at least 35% cross-linking of the fibrin-α-chains after two hours of incubation.

The expressions "plasminogen-activator-inhibitor" and "plasmin-inhibitor" as referred to hereinbefore are to be understood as follows: plasminogen is the inactive precursor of plasmin. Plasmin is a protease which causes the lysis of fibrin. The fibrinolytic activity thus caused can be inhibited either with the help of plasminogen activator inhibitors or plasmin inhibitors or both.

The presence of such inhibitors, as for instance aprotinin, in the tissue adhesive does not influence the adhering properties as such, but prevents the premature lysis of aherences in the body because of a fibrinolytic activity present.

In the tissue adhesive according to the invention plasminogen-activator-inhibitor or plasmin inhibitor has to be present in such a concentration that fibrinolysis is prevented.

In cases where aprotinin is employed as inhibitor it has to be present in an amount of 20 to 2,000 KIU per ml, KIU standing for Kallikrein-inactivator-units.

The ranges of the other inhibitors are as follows:

    ______________________________________                                         C-1-inactivator       0.1-75  U/ml                                             (C.sub.1 -esterase inhibitor)                                                  α.sub.2 -antiplasmin                                                                           0.05-80 U/ml                                             α.sub.2 -macroglobulin                                                                         0.5-90  mg/ml                                            α.sub.1 -antitrypsin                                                                           0.5-90  mg/ml                                            ε-aminocaproic acid                                                                          1-500   mg/ml                                            tranexamic acid       1-300   mg/ml                                            (trans-4-aminomethyl-cyclo-                                                    hexanoic acid)                                                                 ______________________________________                                    

1 U (1 unit) of C1-inactivator and α₂ -antiplasmin, respectively, is the amount contained in 1 ml of fresh pooled citrated human plasma.

As it has been pointed out hereinbefore, the tissue adhesive according to the invention possesses characteristic cross-linking properties, which are determined according to the sodium-dodecyl-sulphate-(SDS)-polyacryl-amide-gel-electrophoresis method, K. Weber and M. Osborn. The Reliability of Molecular Weight Determinations by Dodecyl Sulfate-Polycarylamide Gel Electrophoresis. J. Biol. Chem. 244, 16, pp. 4406-4412 (1969); M. L. Schwartz et al. The Effect of Fibrin Stabilizing Factor on the Subunit Structure of Human Fibrin. J. of Clin. Invest. 50, p. 1506-1513 (1971). After mixing of the tissue adhesive with an equal volume of a solution containing 40 μMoles of CaCl₂ and 15 NIH-units (U.S. National Institute of Health-units) of thrombin per ml, the mixture is incubated at 37° C. The cross-linking degree is determined by gel electrophoresis after stopping the reaction and reductive splitting of the disulphide bridges contained in the proteins by the addition of a mixture of urea, sodium dodecyl sulphate and β-mercaptoethanol.

The invention furthermore comprises a method of producing the tissue adhesive described by starting out from human or animal plasma cryoprecipitate. The method is characterized in that cold-soluble plasma-protein is removed from the cryoprecipitate by a single or repeated treatment with a buffer solution containing sodium citrate, sodium chloride, glycine, glucose and a plasminogen-activator-inhibitor or plasmin inhibitor, and the purified precipitate is dissolved.

Advantageously, the cryoprecipitate has been produced from fresh human or animal plasma frozen at -20° C. When increasing the temperature to 0° to 2° C., the cryoprecipitate is gained and separated by centrifugation. The buffer solution with which the cryoprecipitate is treated has a pH of about 6.0 to 8.0. The cold-soluble plasma protein is separated by centrifugation on maintaining a temperature of 0° to 4° C. The purified precipitate is then washed with the buffer solution until the desired ratio of factor XIII to fibrinogen and the desired proportions of fibrinogen, cold-insoluble globulin and albumin are needed.

The purified precipitate dissolved can be preserved by deepfreezing.

The tissue adhesive according to the invention can be applied universally. It can be used for the seamless connections of human or animal tissue or organs, for sealing wounds and stopping bleedings as well as for the stimulation of wound healing.

Preferred fields of application, in which the tissue adhesive according to the invention can be successfully used, are in the field of ear, nose and throat surgery, oral surgery, dentistry, neurosurgery, plastic surgery, general surgery, abdominal surgery, thorax and vascular surgery, orthopaedics, accident surgery, urology, ophthalmology and gynaecology.

Advantageously, a mixture of thrombin and calcium chloride is added to the adhesive prior to the application of the tissue adhesive according to the invention or is applied onto the tissue to be connected.

The method of the invention will be explained in more detail by the following examples.

EXAMPLE 1

178.5 l of human fresh plasma deepfrozen at -20° C. were heated to +2° C. The cryoprecipitate obtained was separated by centrifugation and treated at +2° C. with 8 l of a buffer solution containing 6.6 g of Na₃ -citrate. 0.2H₂ O, 3.4 g of NaCl, 10.0 g of glycine, 13.0 g of glucose. 1H₂ O and 50,000 KIU of aprotinin per liter, and having a pH of 6.5, and was again centrifuged at +2° C. The treatment with the buffer solution mentioned and the subsequent centrifugation were repeated once. The separated precipitate was liquefied by heating to 37° C.

Aprotinin was present in the product thus obtained in a concentration of approximately 50 KIU per ml. The ratio of fibrinogen to cold-insoluble globulin to albumin in the product was determined to be 86.2:6.7:2.2 This determination was carried out also by the SDS-polyacryl-amide-gel-electrophoresis method, i.e. (a) with a non-reduced sample of the tissue adhesive and (b) with a sample reduced with β-mercaptoethanol. 13.8 units/ml of factor XIII were found and the ratio of factor XIII to fibrinogen, expressed in units of factor XIII per gram of fibrinogen, was 152. Fibrinogen was present in an amount of 90.6 mg/ml.

The determinations were carried out in the following manner: The determination of the factor-XIII-units was performed by means of a cross-linking test in which factor-XIII-free fibrinogen was used as a substrate and the fibrin-cross-linking caused by the addition of the unknown diluted sample served as a measure for the amount of factor XIII contained therein. A corresponding calibration curve was obtained with pooled human citrate plasma, 1 ml plasma containing 1 unit of factor XIII. The protein determinations were carried out by the method according to Kjeldahl.

The cross-linking test according to the SDS-polyacrylamide-gel-electrophoresis method showed complete fibrin-γ-cross-linking after 5 minutes and 68% fibrin-α-cross-linking after 2 hours. The end product was put into final containers and deepfrozen at -20° C. for storage.

EXAMPLE 2

1,2 l of fresh frozen sterile human plasma were thawed at 0° to +4° C. The cryoprecipitate obtained was separated by centrifugation, treated with a sterile, citrated NaCl-glycine-glucose buffer containing 4.4 U/ml C1-inactivator and was again centrifuged. The treatment with the buffer solution mentioned and the subsequent centrifugation were repeated. The tissue adhesive thus obtained was homogenized and liquefied by heating to 37° C., then put into final containers and deepfrozen for storage. In the end product C1-inactivator was present in a concentration of 4 U/ml.

EXAMPLE 3

In the same manner as described in Example 2 above, a tissue adhesive was produced, only that 0.9 U/ml α₂ -antiplasmin, instead of the C1-inactivator, were added to the buffer.

EXAMPLE 4

Again in the same manner as described in Example 2 above, the tissue adhesive was produced by adding 2 mg/ml α₂ -macroglobulin to the buffer, instead of the C1-inactivator.

EXAMPLE 5

The same procedure as described in Example 2 above was employed only that 10 mg/ml α₁ -antitrypsin were added to the buffer instead of the C1-inactivator.

According to a modified method, it is also possible to add the plasminogen-activator-inhibitor or plasmin inhibitor at a later stage, as will be illustrated by the following examples.

EXAMPLE 6

112.5 l of human fresh plasma deepfrozen at -20° C. were heated to +2° C. The resulting cryoprecipitate was separated by centrifugation and washed at +2° C. with 10 ml of a buffer solution containing 6.6 g of Na₃ -citrate. 0.2 H₂ O, 3.4 g of NaCl, 10.0 g of glycine, 13.0 g of glucose. 1H₂ O per liter, and having a pH of 6.5, and was again centrifuged at +2° C. The separated precipitate was liquefied by heating to 37° C. 50 KIU of aprotinin per ml were added to the solution thus obtained, which amount is tantamount to the content of aprotinin in the end product.

In the product thus obtained the ratio of fibrinogen to cold-insoluble globulin to albumin was determined to be 91.0:5.3:1.1. This determination was carried out also by the SDS-polyacrylamide-gel-electrophoresis method, i.e. (a) with a non-reduced sample of the tissue adhesive, and (b) with a sample reduced with β-mercaptoethanol. 15.7 units/ml of factor XIII were found and the ratio of factor XIII to fibrinogen, expressed in units of factor XIII per gram of fibrinogen, was 170. Fibrinogen was present in an amount of 92.1 mg/ml.

The determinations were carried out in the following manner: The determination of the factor-XIII-units was performed by means of a cross-linking test in which factor-XIII-free fibrinogen was used as a substrate and the fibrin-cross-linking caused by the addition of the unknown diluted sample served as a measure for the amount of factor XIII contained therein. A corresponding calibration curve was obtained with pooled human citrate plasma, 1 ml plasma containing 1 unit of factor XIII. The protein determinations were carried out by the method according to Kjeldahl.

The cross-linking test according to the SDS-polyacrylamide-gel-electrophoresis method showed complete fibrin-γ-cross-linking after 5 minutes and 87% fibrin-α-cross-linking after two hours. The end product was put into one-way injection syringes and deepfrozen at -20° C. for storage.

EXAMPLE 7

15.0 l of human fresh plasma deepfrozen at -20° C. were heated to +2° C. The resulting cryoprecipitate was separated by centrifugation and washed at +2° C. with 1,5 l of a citrated NaCl-glycine-glucose-buffer, and was again centrifuged at +2° C. The separated precipitate was liquefied by heating to 37° C. 200 mg of epsilon-aminocaproic acid per ml were added to the solution thus obtained, which amount is tantamount to the content of epsilon-aminocaproic acid in the end product.

EXAMPLE 8

12.0 l of human fresh plasma deepfrozen at -20° C. were heated to +2° C. The cryoprecipitate obtained was separated by centrifugation and treated at +2° C. with 0.6 l of a citrated NaCl-glycine-glucose-buffer, and was again centrifuged at +2° C. The treatment with the buffer solution mentioned and the subsequent centrifugation were repeated once. The precipitate separated was liquefied by heating to 37° C.

100 mg of tranexamic acid per ml were added to the solution thus obtained which amount is tantamount to the content of tranexamic acid in the end product.

The products obtained from the various examples were tested in the above manner as described in Examples 1 and 6 above. The results of these tests are summarized in Table I. There was a complete fibrin-γ-cross-linking after five minutes in the samples according to all of the Examples.

                                      TABLE 1                                      __________________________________________________________________________                                 Ratio of                                                                             Fibrin-α-                                                                              Ratio of                                             Content of                                                                           Fibrinogen:                                                                          Cross-Linking                                                                         Content of                                                                            Factor XIII:                   Example       Approximate                                                                            Fibrinogen                                                                           CIG:  after 2 hours                                                                         Factor XIII                                                                           Fibrinogen                     No.  Inhibitor                                                                               Concentration                                                                          (mg/ml)                                                                              Albumin                                                                              (%)    (U/ml) (U/g fibrinogen)               __________________________________________________________________________     1    aprotinin                                                                               50 KIU/ml                                                                              90.6  86.2:6.7:2.2                                                                         68     13.8   152                            2    C-1-inactivator                                                                         4  U/ml 87.5  88.3:4.2:2.9                                                                         76     14.7   168                            3    α.sub.2 -antiplasmin                                                              0.8                                                                               U/ml 88.3  87.4:5.7:1.7                                                                         72     13.9   157                            4    α.sub.2 -macroglobulin                                                            1.8                                                                               mg/ml                                                                               79.0  82.6:4.7:1.9                                                                         68     12.7   161                            5    α.sub.1 -antitrypsin                                                              9  mg/ml                                                                               89.2  85.7:3.9:2.1                                                                         62     11.9   133                            6    aprotinin                                                                               50 KIU/ml                                                                              92.1  91.0:5.3:1.1                                                                         87     15.7   170                            7    ε-aminocaproic                                                                  200                                                                               mg/ml                                                                               86.8  89.2:4.9:1.5                                                                         78     15.5   179                                 acid                                                                      8    tranexamic                                                                              100                                                                               mg/ml                                                                               89.5  91.3:4.1:1.3                                                                         69     14.3   160                                 acid                                                                      __________________________________________________________________________ 

What we claim is:
 1. A tissue adhesive comprising plasma proteins of human or animal origin, which plasma proteins contain: factor XIII in an amount of at least 7 units/ml, fibrinogen in an amount of at least 70 mg/ml, the ratio of factor XIII to fibrinogen, expressed in units of factor XIII per gram of fibrinogen, amounting to at least 100; cold-insoluble globulin and albumin, the ratio of fibrinogen to cold-insoluble globulin to albumin being 60 to 98:0.5 to 20:0 to 15; plasminogen-activator inhibitor or plasmin inhibitor in an amount of 20 to 2,000 KIU per ml, wherein the tissue adhesive as determined according to the SDS-polyacrylamide-gel-electrophoresis method is capable of complete cross-linking of the fibrin-γ-chains after 3 to 5 minutes of incubation and of at least 35% cross-linking of the fibrin-α-chains after two hours of incubation.
 2. A method of producing a tissue adhesive as described in claim 1, which comprises:(a) thawing deepfrozen fresh plasma to obtain a cryoprecipitate and a soluble plasma protein; (b) separating the cryoprecipitate from the soluble plasma protein; (c) washing the thus obtained cryoprecipitate at about 2° C. with a buffer solution having a pH of about 6.5 and containing sodium citrate, sodium chloride, glycine, glucose and a plasminogen-activator inhibitor or a plasmin inhibitor; (d) heating the washed precipitate to about 37° C. to obtain a liquid tissue adhesive; and (e) deepfreezing the liquid tissue adhesive until ready to use.
 3. A method of producing a tissue adhesive as described in claim 1, which comprises the steps of:(a) thawing deepfrozen fresh plasma to obtain a cryoprecipitate and soluble plasma protein; (b) separating the cryoprecipitate from the soluble plasma protein; (c) washing the thus obtained cryoprecipitate at about 2° C. with a buffer solution having a pH of about 6.5 and containing sodium citrate, sodium chloride, glycine and glucose; (d) heating the washed precipitate to about 37° C. to obtain a liquid tissue adhesive; (e) adding a plasminogen-activator inhibitor or a plasmin inhibitor and (f) deepfreezing the liquid tissue adhesive until ready to use.
 4. A method of tissue or organ adhesion in mammals which comprises applying onto the organ or tissue an amount sufficient to function as an adhesive of the adhesive as described in claim 1 and a mixture of thrombin and calcium chloride.
 5. The method as described in claim 4, wherein the mixture of thrombin and calcium chloride is added to the adhesive prior to applying the tissue adhesive to the tissue or organ.
 6. A tissue adhesive comprising plasma proteins of human or animal origin, which plasma proteins contain: factor XIII in an amount of at least 7 units/ml, fibrinogen in an amount of at least 70 mg/ml, the ratio of factor XIII to fibrinogen, expressed in units of factor XIII per gram of fibrinogen, amounting to at least 100; cold-insoluble globulin and albumin, the ratio of fibrinogen to cold-insoluble globulin to albumin being 60 to 98:0.5 to 20:0 to 15; plasminogen-activator-inhibitor or plasmin inhibitor selected from the group consisting of C1-inactivator, α₂ -antiplasmin, α₂ -macroglobulin, α₁ -antitrypsin, epsilonaminocaproic acid and tranexamic acid, the concentration being such that fibrinolysis is prevented, wherein the tissue adhesive as determined according to the SDS-polyacrylamide-gel-electrophoresis method is capable of complete cross-linking of the fibrin-γ-chains after 3 to 5 minutes of incubation and of at least 35% cross-linking of the fibrin-α-chains after two hours of incubation.
 7. A tissue adhesive as described in claim 6 wherein C1-inactivator in an amount of 0.1 to 75 U/ml is used.
 8. A tissue adhesive as described in claim 2 wherein α₂ -antiplasmin in an amount of 0.05 to 80 U/ml is used.
 9. A tissue adhesive as described in claim 6 wherein α₂ -macroglobulin in an amount of 0.5 to 90 mg/ml is used.
 10. A tissue adhesive as described in claim 6 wherein α₁ -antitrypsin in an amount of 0.5 to 90 mg/ml is used.
 11. A tissue adhesive as described in claim 6 wherein epsilon-aminocaproic acid in an amount of 1 to 500 mg/ml is used.
 12. A tissue adhesive as described in claim 6 wherein tranexamic acid in an amount of 1 to 300 mg/ml is used. 